Damage to connections within the adult Central Nervous System (CNS) by injury or disease is often irreparable. To design therapies to repair CNS damage requires a detailed understanding of the cellular mechanisms underlying CNS development. As the brain matures, neurons migrate to their proper positions within the brain and elaborate processes that are guided to their targets to form proper connections. Both initial formation of growth cone-tipped neurites, and subsequent guided locomotion of axonal growth cones, require F-actin and microtubule (MT) dynamics. F-actin:MT interactions likely play key roles in both of these processes. However, the molecular mechanisms that mediate such interactions, and how these interactions drive neuritogenesis and axon guidance, are not understood. Ena/VASP proteins function in growth cone guidance by controlling actin cytoskeleton dynamics. Using a combination of mouse genetics, primary cell culture, live cell imaging and electron microscopy, my lab found that Ena/VASP-deficient cortical neurons fail to form filopodia, finger-like processes comprised of bundled F-actin. Furthermore, we found that cortical neurons devoid of filopodia fail to form neurites, and exhibit altered microtubule dynamics. Restoration of filopodia in Ena/VASP mutant cortical neurons also rescues neurite initiation. Preliminary data indicate that Ena/VASP-deficient sensory neurons form axons, but exhibit striking guidance defects. We will use sensory neuron preparations from Ena/VASP deficient animals to test our working hypothesis is that Ena/VASP- dependent filopodia formation enables interactions between MTs and actin bundles that are required to receive both attractive and repulsive cues. Additional new data indicate that Ena/VASP proteins may act to coordinate F-actin:MT interactions, and that the TRIM9 protein interacts with both Ena/VASP and microtubules; TRIM9 is also implicated in the control of axon navigation. Furthermore, a network of proteins, including Ena/VASP, is likely regulated by Lamellipodin, a molecule that integrates signals generated by cell-surface receptors for axonal guidance factors. Collectively, these data lead us to hypothesize that Ena/VASP proteins participate in protein networks that play key roles in F-actin: MTs interactions, and are in turn linked to signaling pathways controlled by guidance receptors. Our long-term goal is to understand how neurons integrate environmental cues to orchestrate changes in their morphology and movement necessary to establish a functional nervous system. A better understanding of the mechanistic basis of neurite formation and axon guidance will provide fundamental insight into how connections in the nervous system are established and how they are remodeled during plasticity. The results of our research plan should be of great value to the development of therapeutic approaches to repair these connections subsequent to disease or injury. PUBLIC HEALTH RELEVANCE: Damage to the nerves that form connections within the adult brain and spinal column by injury or disease is often irreparable. We seek a comprehensive understanding of how nerve fibers form and are guided to their targets initially during fetal development expecting that this will help us learn to repair damage to the brain.